Project Summary Hypertrophic cardiomyopathy (HCM) is a highly prevalent hereditary cardiac disease with over 1400 mutations identified in 11 genes encoding the protein constituents of sarcomere, the basic contractile units of muscle. However, the molecular mechanisms by which mutations in sarcomeric proteins lead to cardiac hypertrophy and failure remain poorly understood. Sarcomeres are composed of myofilaments flanked by dense protein structures called Z-discs, playing critical roles in controlling contraction and mediating signaling pathways. Moreover, recent evidence has suggested that sarcomeric protein post-translational modifications (PTMs) play essential roles in the modulation of contractile function. Our preliminary data convincingly show that the PTMs of sarcomeric proteins, including multiple key myofilament and Z-disc proteins, are altered in the myocardium of HCM patients as compared to donor hearts. Changes in the expression of myofilament protein isoforms were also detected in HCM patient myocardium. Intriguingly, we have observed consistent alterations in sarcomeric protein PTMs and isoform expression regardless of the mutation types. Thus, we hypothesize that different HCM- causing mutations activate convergent hypertrophic signaling pathways, yielding comparable alterations in PTMs and protein isoforms of the sarcomeric proteins that cause abnormal contractile function and pathological hypertrophy. To test our hypothesis, we will employ a highly interdisciplinary approach featuring top-down proteomics, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), cardiac tissue engineering, human patient heart tissue samples, and functional studies to delineate the underlying mechanisms of HCM. Our recently established top-down proteomics platform represents a comprehensive tool to detect and characterize HCM-related changes in the sarcomeric proteome with high reproducibility and throughput. Recently, we have made a major methodologic breakthrough that allows us to perform top-down proteomic analysis of minimal amounts of biopsy tissue, as well as iPSC-CMs cultured in either a 2D monolayer or 3D engineered cardiac tissue (ECT). In addition, biochemical analyses will be performed to assess changes in intracellular signaling pathways. Furthermore, we will perform functional measurements to relate sarcomeric protein PTM alterations and maladaptive signaling to contractile dysfunction. We aim to determine: 1) the changes in sarcomeric protein PTMs, key hypertrophic signaling pathways, and mechanical properties in myocardial tissue from HCM patients; 2) the early molecular alterations and functional consequences of different sarcomeric protein mutations in HCM patient-specific iPSC-CMs; 3) the specific molecular alterations and functional consequences in iPSC-CM- ECT from multiple HCM patients carrying the same mutation. This application, enabled by cutting-edge technology, is highly significant with a strong scientific premise and a novel hypothesis. The proposed research has direct translational potential and will lead to an in-depth understanding of the underlying mechanisms in HCM.